Hepatitis B virus causes chronic infections in approximately 350 million people worldwide. All are at significantly increased risk of developing hepatocellular carcinoma. Although virus replication can be blocked by therapy with nucleoside analogs, infected hepatocytes are not cured, because covalently closed circular DNA (CCC DNA), the template for transcription of viral RNAs persists in infected cells. So far, a therapeuti strategy to functionally inactivate or even destroy CCC DNA within infected hepatocytes is lacking. We propose to use transcription activator-like effector nucleases (TALENs) specific for CCC DNA to cleave and mutate CCC DNA in regions essential for viral DNA replication. Hence, the goal of this application is to obtain proof of principle for the idea that an infected hepatocye can be cured of HBV through inactivation and possibly destruction of CCC DNA. Preliminary experiments from our laboratory already demonstrated that CCC DNA is indeed susceptible to cleavage by TALENs. The purpose of this proposal is to investigate the potential of TALENs to functionally inactivate CCC DNA by insertion/deletion of bases during the repair reaction, or to even destroy CCC DNA and to determine whether TALEN-based antiviral therapy could lead to the cure of infected cells. As a model for HBV, we will be using duck hepatitis B virus (DHBV) expressed in hepatoma cells and primary hepatocyte cultures that permit all steps of viral DNA replication including the formation of CCC DNA. The goals of this application will be achieved through the following two aims: Aim 1. Construction and validation of TALENs specific for DHBV. The purpose of this aim is i) to construct TALENs against three targets on DHBV required for DNA replication, ii) to measure the ability of each TALEN to cleave CCC DNA, iii) to determine the effects of cleavage on CCC DNA stability, and iv) to investigate whether cleavage by TALENs leads to integration of CCC DNA into chromosomal DNA. Aim 2. To determine the antiviral activity of TALENs. The purpose of this aim is to study the effect of TALEN-based therapy on infection of dividing hepatoma cells and non-dividing primary duck hepatocytes. We will investigate whether TALEN therapy leads to a time-dependent reduction in CCC DNA levels per cell and determine not only if CCC DNA can be functionally inactivated in non-dividing cells, but also if it can be destroyed without a need for cell death. Furthermore, we will determine whether TALEN therapy can protect hepatocytes from de novo infection.